1. Field of the Invention
The present invention relates generally to the field of biochemical endocrinology of corticotropin releasing hormone receptors. More specifically, the present invention relates to the identification of alternative splicing variants of human and mouse corticotropin releasing hormone receptor-1.
2. Description of the Related Art
Corticotropin releasing hormone (CRH, also known a s corticotropin releasing factor or CRF) is the most proximal element of the hypothalamic-pituitary-adrenal (HPA) axis that coordinates the complex array of behavioral, autonomic, endocrine and immune responses to stress. The peptide signal is translated into phenotypic effects through interaction with G protein-coupled, membrane-bound CRH receptors (1). Two subtypes of receptors, Type 1 (CRH-R1) and Type 2 (CRH-R2), have been characterized in humans (2, 3), rats (4–6), mice (7–9) and Xenopus (10). Most recently a third subtype, CRH-R3, has been identified in catfish (11).
CRH-R1 is a protein with 98% sequence homology among different mammalian species and approximately 30% homology with receptors for the gut-brain family of neuropeptides (1, 12, 13). The human CRH-R1 gene contains 14 exons (14). Four alternatively spliced CRH-R1 transcripts have been identified in humans. These are CRH-R1α, in which exon 6 is spliced out to generate a 13-exon transcript that produces a 415-amino acid protein (2); CRH-R1β, which contains all 14 exons to produce a 444-amino acid protein (2); a CRH-R1c isoform, where exons 3 and 6 are spliced out to generate a 12-exon transcript producing a 375-amino acid protein (15); and an CRH-R1d isoform, where exons 6 and 13 are spliced out to produce a 401-amino acid protein (16).
CRH-R1 isoforms have different affinity for receptor ligands, resulting in differences in coupling of the isoforms to cAMP production signaling. The major ligand-binding determinant of mammalian CRH-R1 has been mapped to its first extracellular domain (17). This domain is encoded by exons 1–4 of CRH-R1. Exon 3 contains two regions that are critical for high-affinity ligand binding; thus, mutations in this region abolish CRH binding (18). The CRH-R1c isoform, which lacks exon 3, should therefore have a decreased CRH binding capacity. A 29 amino acid insert corresponding to exon 6 of CRH-R1β has also been reported to decrease binding affinity as well as coupling of the receptor to G proteins (19). A CRH-R1d isoform lacking exon 13 has been recently cloned from human myometrium (16). This isoform is poorly coupled to G proteins. Thus, it appears that CRH-R1α is the most efficient receptor isoform in transducing a CRH signal into cAMP-mediated pathways, while other isoforms either have a poor ligand-binding capacity or are poorly coupled to cAMP production. Because a spectrum of receptor isoforms expressed by a cell can determine its response to a ligand, full molecular characterization of CRH-R1 transcripts is necessary in order to understand the pleiotropic role of CRH.
Skin, the largest body organ, maintains internal homeostasis by serving as a barrier between the external environment and the internal milieu. Being continuously exposed to noxious stimuli of varying intensities, including solar radiation, thermal energy and biological agents, the skin requires a highly localized and precise mechanism for dealing with the immediacy of these interactions (20, 21). Analogous to the central response to stress centered on the HPA axis, it was proposed that similar mediators could activate peripheral responses to stress with a CRH-based signaling system playing a major regulatory role (22–24).
Both CRH and urocortin are produced in human and rodent skin, accompanied by the expression of functional CRH-R1 (21–26). It has been proposed that the flow of information involving cutaneous CRH peptides could be arranged hierarchically, from CRH through CRH-R1 to the activation of POMC peptide production and corresponding activation of the respective receptors for these peptides (22, 24). Alternatively, they could act directly through CRH-R1 activated pathways to regulate epidermal integrity, barrier function, immunomodulation, dermal vascular function, and hair growth and pigmentation (20, 22, 24). Such functional diversity requires specific molecular mediators, and functional selectivity could be achieved through differential expression of CRH-R1 isoforms.
The prior art is deficient in a full molecular characterization of CRH-R1 isoform expression for understanding the pleiotropic effects of CRH. The present invention fulfills this long-standing need and desire in the art.